Steinernema goweni, San-Blas, Ernesto, Morales-Montero, Patricia, Portillo, Edgar, Nermuť, Jiří & Puza, Vladimir, 2016

San-Blas, Ernesto, Morales-Montero, Patricia, Portillo, Edgar, Nermuť, Jiří & Puza, Vladimir, 2016, Steinernema goweni n. sp. (Rhabditida: Steinernematidae), a new entomopathogenic nematode from Zulia State, Venezuela, Zootaxa 4067 (2), pp. 200-214 : 202-213

Steinernema goweni * n. sp.

( Figs. 1–5 )

*specific epithet after Dr. Simon Gowen a British nematologist.

Description. Male, first generation. Measurements of 20 males are given in Table 1 . Body curved ventrally posteriorly. Lateral field not seen under light microscopy. Head round and continuous with body ( Fig. 1 A). Face with six labial and four cephalic papillae, gently tapering anteriorly. Amphidial apertures visible with SEM and located posterior to the lateral labial papillae ( Fig. 5 A). Stoma shallow, cuticularised. Excretory pore anterior to nerve ring. Pharynx muscular, cylindrical procorpus, swollen metacorpus, narrow isthmus and distinct basal bulb. Nerve ring surrounding isthmus or anterior part of the basal bulb. Gonad monorchic, testis reflexed comprising germinal zone, growth zone and vas deferens. Males in both generation possess 25 or 27 (in 15% of the specimens) genital papillae comprising 12 or 13 pairs and a single ventral papilla located anterior to cloacal opening. Specimens with twenty five genital papillae visible with light microscopy (12 pairs and one single ventral precloacal papillae) including six subventral precloacal pairs, one pair adanal, one pair cloacal, one pair lateral, one pair postcloacal subdorsal and two subterminal ( Fig. 5 B). Specimens with 27 genital papillae have an extra lateral pair anterior to the cloaca (at the same level than the fourth precloacal papilla) ( Fig. 5 C). Spicules paired, dark brown in colour, head (manubrium) round dorsally directed, calomus (shaft) short, with a prominent ventral arch ( Figs. 1 B, 3D, 3D1). Two visible internal ribs and tapering gradually posteriorly to a point. Velum always present. Gubernaculum boat shaped in lateral view. Cuneus Y-shaped in ventral view ( Figs. 1 C, 3D2). Tail conoid. Phasmids always visible at SEM view and an unknown pore-like structure can be seen at the right of tail tip at SEM view in different specimens ( Fig. 5 D). Mucron absent.

Male, second generation. Similar to first generation males but smaller in many features and proportions ( Table 1 ). Gubernaculum boat shaped but, with posterior part variable between tapered, squared and hooked-like ventrally ( Figs. 1 D to 1G)

Female, first generation. Measurements of 20 female are given in Table 1 . Lateral field not seen under light microscopy. Head round, continuous with body ( Fig. 2 A). Six labial and 4 cephalic papillae present. Amphidial apertures lateral and at cephalic papillae level. Procorpus cylindrical, metacorpus swollen and isthmus surrounded by nerve ring. Basal bulb distinct. Excretory pore prominent, anterior to nerve ring. Ovaries amphidelphic and reflexed, vulva a transverse slit, around the middle of the body (55%) slightly protruding or not, epitygmata present and pointed posteriorly, varying in size ( Figs. 2 D to 2F), oviduct well developed, spermatheca present and filled with 1 to 4 spermatozoa ( Figs. 1 B, 2G). Post-anal swelling not present or inconspicuous in 20% of specimens ( Figs. 2 B, 2C). Phasmids visible under SEM ( Fig. 4 E). Tail short, with a mucron-like terminus in some specimens ( Figs. 1 B, 1C, 4D).

TABLE 2. Cοmparative mοrphοmetrics οf infective ϳuveniles οf Steinernema goweni n. sp. and related Steinernema spp. frοm the " bicornutum " grοup (in descending οrder οf bοdy length). All measurements are in μm and in the fοrm∶ mean (range).

= maximum bοdy width; EP = distance frοm anteriοr end tο excretοry pοre; NR = distance frοm anteriοr end tο nerve ring; ES = esοphagus length; Τ = tail length; D% = EP⁄ES) × 100; E% = ⁄Τ) × 100

Female, second generation. Similar to first generation females but smaller in many features and proportions ( Table 1 ).

Infective juvenile. Measurements of 20 infective juveniles are given in Table 1 . Body elongated. Labial region smooth, continuous with body. Cuticule transversely striated. Exsheathed IJ with two horn-like structures on labial region ( Fig. 1 J), very distinct by light microscopy and SEM, four cephalic papillae and amphidial apertures (lateral and located posterior to the horn-like structures) visible with SEM ( Fig. 4 A). Stoma closed. Excretory pore anterior to nerve ring. Procorpus narrow, metacarpus a slightly swollen, distinct but small basal bulb with a distinct valve.

Isthmus present, surrounded by nerve ring. Lateral field with eight ridges evenly spaced at mid-body; beginning with a single line, becoming two ridges splitting into four, then in six and eight (of which submarginal ridges are less distinct), at mid-body ( Fig. 4 B). At this point, 3rd and 6th ridges becoming wider than others which are still present when ridges reduce to six and then forming the last two ridges which continue almost to the tail tip. Lateral field formula 2,4,6,8,6,2. Hyaline portion occupying 57 (45–66) % of tail length ( Fig. 1 I). Phasmids always visible in SEM ( Fig. 4 C) located at one-third of the tail length from the anus.

Diagnosis and relationships. Based on its morphology, S. goweni n. sp. belongs to the “ bicornutum ” group in having two horn-like structures on the labial region of the IJ, and differs from the other eight recognised species in this group by a combination of morphological, morphometric and DNA characters.

Steinernema goweni n. sp. is characterised by morphometrics of its IJ ( Table 1 ), with body length of 640 (531– 719) µm, pharynx length of 119 (109–126) µm, a tail of 67 (59–89) µm, c ratio = 9 (6–11) and E% = 77 (48–94). Males of the first generation can be recognised by the shape and size of their spicules 55 (50–57) µm long and gubernaculum 35 (30–40) µm long, and their %D value 42 (28–59), which is the lowest within the “ bicornutum ” group ( Table 3 ). The number of genital papillae in males, namely 27 (13 pairs + 1) papillae in 15% of their specimens is distinctive of S. goweni n. sp. As too the male phasmids, which are very distinct in S. goweni n. sp. but have not been reported within the “ bicornutum ” group.

Males of S. goweni n. sp. can be distinguished from S. riobrave by a higher number of genital papillae (25 or 27 from the former vs. 23 from the latter), shorter spicules (55 (50–57) µm vs. 67 (62–75) µm) and gubernaculum (35 (30–40) µm vs. 51(47–56) µm), and lower MBD (100 (85–115) µm vs. 133 (116–159) µm) and GS% (64 (49– 79) vs. 76). Phasmids are prominent in SEM view and have not been reported in S. riobrave . Furthermore, the spicule shape is different in both species; S. goweni n. sp. possesses a rounded manubrium dorsally directed and a velum, but S. riobrave has an elongated, broad and somewhat angular-shaped manubrium and lacks a velum. Infective juveniles of S. goweni n. sp. have a longer tail (67 (59–89) µm vs. 54 (46–59) µm) and a lower %E value (77 (48–94) vs. (105 (93–111)). Females in both generations of S. goweni n. sp. possess epitygmata, whereas females of S. riobrave have no epitygmata. Post anal swelling in S. goweni n. sp. is absent or inconspicuous in 20% of the specimens but second generation females of S. riobrave present a prominent post anal swelling.

Steinernema goweni n. sp. can be distinguished from S. papillatum males by a higher number of genital papillae (25 or 27 vs. 23), the presence of a cloacal papilla, and phasmids. Infective juveniles of both species differ in their length of the tails (67 (59–89) µm vs. 54 (40–78) µm) and the presence of prominent phasmids in S. goweni n. sp. which can be observed by SEM. First generation females of S. goweni n. sp. are shorter than females of S. papillatum (2861 (1836–4479) µm vs. (6728 (3333–12568) µm and have a shorter distance from the anterior end to the excretory pore, 58 (40–88) µm vs. 101 (66–129) µm, and a shorter pharynx (164 (135–188) µm vs. (194 (160– 1242) µm. Post-anal swelling in S. goweni n. sp. is absent or inconspicuous in 20% of the specimens but females of S. papillatum present a prominent post anal swelling in 50% of the specimens.

Steinernema goweni n. sp. can be distinguished from S. bicornutum males by the position of the cloacal papilla (only present in S. goweni n. sp.). The second generation males of S. goweni n. sp. possess spicules with velum and lacks mucron whereas S. bicornutum have spicules without velum and presents mucron. Both species also differ in in the length of spicules (55 (50–57) µm vs. 65 (53–70) µm), gubernaculum (35 (30–40) µm vs. 48 (38–50) µm) and by the SW% value (146 (105–208) µm vs. 222 (218–226) µm). The IJs of S. goweni n. sp. can be recognised from S. bicornutum by having a lower body length (640 (531–719) µm vs. 769 (648–873) µm), by a shorter distance from head to excretory pore (51 (32–58) µm vs. 61 (53–65) µm) and the presence of phasmids.

Males of S. goweni n. sp. can be separated from S. yirgalemense by the length of spicules (55 (50–57) µm vs.

64 (51–77) µm), and gubernaculum (35(30–40) µm vs. 48 (42–54) µm), by absence of mucron in both generations and the presence of the phasmids (not reported in S. yirgalemense ). Second generation females of S. goweni n. sp. can be distinguished from S. yirgalemense by the absence of a mucro.

Steinernema goweni n. sp. males can be distinguished from S. ceratophorum by having smaller spicules (55 (50–57) µm vs. 71 (54–90) µm) and their shape; the manubrium of S. goweni n. sp. is rounded vs. somewhat angular in the latter species. Genital papillae differ in numbers (25 or 27 vs. 23 in S. ceratophorum ) and by the presence of phasmids (not reported in S. ceratophorum ). The IJs of the new species are shorter than IJs than those of S. ceratophorum (640 (531–719) µm vs. 706 (591–800) µm) and have distinct phasmids (not reported in S. ceratophorum ). Females of S. goweni n. sp. exhibit epitygmata, that are absent in S. ceratophorum .

The number of genital papillae in males of S. goweni n. sp. is greater than S. pakistanense (25 or 27 vs. 23). The spicules length of S. goweni n. sp. are shorter than those of S. pakistanense (55 (50–57) µm vs. 68 (62–73) µm) and shape also differ between these two species. IJs from S. goweni n. sp. have longer tails (67 (59–89) µm vs. 58 (53–62) µm.

Males of S. goweni n. sp. can be also differentiated from S. abbasi males by the lower values of SL (55 (50– 57) µm vs. 65 (57–74) µm), GL (35 (30–40) µm vs. (45 (33–50) µm), %D (42 (28–59) vs. 60 (51–68)) and %GS (64 (49–79) vs. 70 (58–85)). IJs of S. goweni n. sp. are longer (640 531–719) µm vs. 541 (496–579) µm). Steinernema goweni n. sp. differs from S. bifurcatum by the presence of a gubernaculum that is bifurcated at both proximal and distal ends. Steinernema goweni n. sp. IJ can be recognized from S. bifurcatum by higher body length (640 ((531–719) µm vs. 521 (460–590) µm). The spicules are different in size (55 (50–57) µm vs. 69 (60–85) µm). Steinernema bifurcatum males also differ from S. goweni n. sp. by presence of mucro.

Type host and locality. The host of this nematode in nature is unknown. Steinernema goweni n. sp. was collected from a sandy loam soil (10°16’53’’N; 70°50’58’’W) close to “El Venado” (Baralt Municipality) a little town that is located in the eastern coast of Maracaibo estuary, in Zulia State ( Venezuela).

Type material. Holotype male (first generation), five paratype males (first generation), five paratype females (first generation) and two slides containing six paratype IJs were deposited in The University of California Riverside Nematode Collection ( UCRNC), CA, USA (Accession numbers: from 31466 to 31478). The rest of the paratypes material remains in the Laboratorio de Protección Vegetal (LPV), at Instituto Venezolano de Investigaciones Científicas, Maracaibo, Venezuela. Living material is available at LPV and at the Institute of Entomology, Branišovská, České Budějovice, Czech Republic.

Molecular characterisation and Phylogenetic analysis. Gel electrophoresis revealed a PCR-product of 964 bp for the ITS-region, comprising the transcribed spacer regions ITS1 and ITS2, the 5.8S rRNA gene and flanking regions of the 18S rRNA and 28S rRNA gene. No sign of the intra-individual variability in the ITS sequence was observed.

Steinernema goweni n. sp. is separated by 148–250 bp from other species in “ bicornutum ” group ( Table 4 ). The D2 and D3 expansion fragments of the 28S rRNA gene, generated by PCR, had a 837 bp length and is separated by 51–103 bp from other species in “ bicornutum ” group ( Table 4 ).

Phylogenetic analyses of the “ bicornutum ” group based on both ITS and D2D3 regions showed a clear separation of S. goweni n. sp. from other species in the “ bicornutum ” group ( Figs. 6 and 7 ). In both analyses S. goweni n. sp. formed a strongly supported group of American species with S. riobrave but the higher-level topology differed depending on the marker used. In agreement with San-Blas et al. (2015a), in the ITS tree, S. goweni – S. papillatum – S. riobrave formed a group with S. bicornutum – S. ceratophorum and S. abbasi – S. yirgalemense are sister to this clade. In the D2D3 tree, S. goweni – S. papillatum – S. riobrave grouped with S. abbasi – S. bifurcatum – S. yirgalemense . In general, molecular data confirm the status of S. goweni n. sp. as a new species according to the phylogenetic and evolutionary species concept ( Adams, 1998).

Figures

FIGURE 1. Male and infective juveniles of Steinernema goweni n. sp. A) male of first generation in toto; B) spicule of first generation male; C) gubernaculum of first generation male in ventral view; D, E, F, G) different gubernacula shapes in second generation males; H) head and pharyngeal region of infective juvenile; I) tail of infective juvenile. Scale bars 20 µm

FIGURE 2. Females of Steinernema goweni n. sp. A) head and pharyngeal region of first generation female; B, C) different female tails; D, E, F) vulvae showing different epitygmata G) reproductive system section showing uterus, spermatheca, oviduct and part of the ovary. Scale bars 20 µm

FIGURE 3. Microphotography of female and male of Steinernema goweni n. sp. A) head and pharyngeal region of first generation female; B) detail of female reproductive system, (u = uterus, sp = spermatheca; sz = spermatozoa, ov = oviduct); C) female tail; D) tail of first generation male (insert 1 = spicule, 2 = gubernaculum in ventral view). Scale bars 20 µm

FIGURE 4. Scanning electron microscope photography of infective juvenile and female of Steinernema goweni n. sp. A) head of infective juvenile showing the “ horn-like ” structures on the labial region; B) lateral field arrangement at mid body of infective juvenile; C) tail of infective juvenile showing phasmids (arrow); D) female terminus E) female terminus showing phasmids (arrow). Scale bars 10 µm

FIGURE 5. Scanning electron microscope photography of male of Steinernema goweni n. sp. A) head showing amphids (arrow); B) posterior region showing the genital papillae distribution (vp = ventral papilla); C) posterior region showing the genital papillae distribution showing an extra lateral papilla (# 13); D) male terminus showing phasmids (ph) and pore (arrow). Scale bars 10 µm.

FIGURE 6. Phylogenetic relationships in the “ bicornutum ” group of Steinernema based on analysis of ITS rDNA regions. Steinernema scapterisci and S. nepalense were used as outgroup taxa. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (10000 replicates) are shown next to the branches. Branch lengths indicate evolutionary distances and are expressed in the units of number of base differences per site.

FIGURE 7. Phylogenetic relationships in the “ bicornutum ” group of Steinernema based on analysis of D 2 D 3 regions of the 28 S rDNA. Steinernema scapterisci and S. nepalense were used as outgroup taxa. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (10000 replicates) are shown next to the branches. Branch lengths indicate evolutionary distances and are expressed in the units of number of base differences per site.

Tables

TABLE 1. Mοrphοmetrics οf Steinernema goweni n. sp. All measurements are in μm and in the fοrm ∶ mean ± s. d. (range) οtherwise is nοtated in the rοws.

Character	First generatiοn		Secοnd generatiοn	Infective ϳuveniles
	Male	Female	Male Female
	Hοlοtype
N	20	20	20 20	20

TABLE 3. Comparative morphometrics of first generation males of Steinernema goweni n. sp. and related Steinernema spp. from the “ bicornutum ” group (in descending order of body length). All measurements are in ìm and in the form: mean (range), otherwise is stated.

S. ceratophorum	71 (54–90)	40 (25–45)	146 (104–185)	51 (33–65)	140 (100–200)	60 (40–80)	Jian et al., 1997
S. pakistanense	68 (62–73)	41 (36–45)	102 (80–128)	60 (50–60)	180 (100–220)	60 (50–60)	Shahina et al., 2001
S. goweni n. sp.	55 (50–57)	35 (30–40)	100 (85–115)	42 (28–59)	146 (105–208)	64 (49–79)
S. papillatum	52 (42–62)	31 (23–36)	69 (54–87)	54 (43–65)	156 (125–194)	59 (48–70)	San-Blas et al., 2015
S. yirgalemense	64 (51–77)	48 (42–54)	112 (97–138)	58 (50–66)	171 (121–213)	74 (65–85)	Nguyen et al., 2004
S. riobrave	67 (62.5–75.0)	51 (47.5–56.2)	133 (116–159)	71 (60–80)	114	76	Cabanillas et al., 1994
S. abbasi	65 (57–74)	45 (33–50)	87 (82–98)	60 (51–68)	156 (187–197)	70 (58–85)	Elawad et al., 1997
S. bifurcatum	69 (60–85)	39 (30–49)	108 (85–117)	58 (50–66)	138 (120–170)	59 (51–79)	Fayyaz et al., 2014

TABLE 4. Pairwise distances of ITS and D 2 D 3 regions between species of the “ bicornutum ” group (below diagonal: percentage similarity, above diagonal: total character differences). Data for Steinernema goweni n. sp. in bold.

ITS region	gow	pap	rio	bic	cer	abb	yir	pak	bif
KR781038 S. goweni n. sp.		148	166	182	165	192	210	250	234
KJ913707 S. papillatum	83.0		125	193	170	184	209	254	236
DQ835613 S. riobrave	82.2	85.6		195	188	210	221	259	236
AY171279 S. bicornutum	75.6	73.8	73.6		108	204	218	244	243
AY230165 S. ceratophorum	81.8	79.6	80.2	85.7		178	198	237	213
AY230158 S. abbasi	76.3	75.3	73.7	71.9	77.7		179	242	233
AY748450 S. yirgalemense	76.5	74.7	76.5	69.6	78.6	77.3		260	245
AY230181 S. pakistanense	72.8	70.2	73.0	66.3	75.7	70.0	72.5		12
JX989267 S. bifurcatum	70.7	68.6	70.1	66.6	72.1	70.3	68.5	98.5
D2D3 region	gow	pap	rio	bic	cer	yir	abb	bif
KR781039 S. goweni n. sp.		51	52	67	73	52	91	103
KJ913708 S. papillatum	93.9		41	80	86	60	97	109
AF331893 S. riobrave	93.6	95.0		89	96	55	109	141
AF331904 S. bicornutum	91.8	90.2	89.7		36	50	87	120
AF331888 S. ceratophorum	91.0	89.4	89.0	95.9		55	93	126
AY748451 S. yirgalemense	91.2	89.8	90.7	91.6	90.7		32	32
AF331890 S. abbasi	88.8	88.1	87.4	90.0	89.3	94.6		42
JQ838179 S. bifurcatum	87.7	86.9	83.6	86.1	85.4	94.6	95.1